Tyrosine phosphorylation is a functional feature of many vital signal- transduction pathways in the cell. The function of phosphorylation rests on basic physical principles, and thus a complete understanding of this function requires structural information, and preferably, energetic analysis. As such, in this project, the control of protein- protein associations through tyrosine phosphorylation is to be investigated by NMR and computational studies. Our earlier work lead to a proposed mechanism by which tyrosine phosphorylation mediates against protein-protein association through a conformational destabilization. The studies described here test the proposed physical basis for destabilization by making structural modification related to the interaction of erythrocyte band 3 with aldolase, and by evaluating structural effects of phosphorylation on band 3 itself. In addition, studies will be initiated to determine if the same model would hold for the interaction between the microtubule-associated protein, tau, and alpha-tubulin. The tau/tubulin interaction is required for the fundamental process of microtubule assembly, and a similar phosphorylation control as that for the association of band 3 with aldolase appears to be involved. A second goal of the proposed research is to begin to describe the structural elements involved in the recognition of a non-receptor tyrosine kinase for its substrate. We will investigate Lyn kinase and Ig-alpha, a coreceptor of the B-cell antigen receptor which is phosphorylated by Lyn when antigen engages the receptor. Direct NMR methods and exchange-transferred nuclear Overhauser experiments will be employed to characterize 3-dimensional structure. In addition to simulated annealing, conformational searching will be done by molecular dynamics in four spatial dimensions, by Monte Carlo minimization, and by genetic algorithms. Conformational heterogeneity in the peptide was found in previous work, and will be considered in this project by comparison of observed NOE intensities with those calculated using a complete rate matrix including exchange effects, and taking into account conformational averaging. The proposed research relates to health by developing principles about tyrosine phosphorylation control in cell signaling. The results will be relevant to the design of drugs that act against cancer and protozoal parasites.